Modified Project Summary/Abstract Section Copper (Cu) is an essential micronutrient that acts as a catalytic cofactor for a number of enzymes including cytochrome c oxidase (CcO), a critical enzyme required for mitochondrial energy production. Cu delivery to CcO is a complex process requiring multiple proteins, and loss-of-function mutations in several of the genes encoding these proteins cause fatal infantile disorders. Currently, no effective therapy exists for these fatal disorders. This is due, in part, to our incomplete understanding of how Cu is trafficked to the mitochondria and is delivered to the Cu-containing subunits of CcO. To address this gap in our knowledge, we use complementary approaches involving yeast genetics and biochemistry to discover and characterize the novel mitochondrial proteins that facilitate Cu delivery to CcO. Using these approaches, we have found the evolutionarily conserved mitochondrial proteins Coa4 and Coa6 as new members of the mitochondrial Cu delivery pathways to CcO. Now, we aim to define the biochemical functions of Coa4 and Coa6 in the Cu delivery process. Our preliminary findings, including the structure of human COA6, have led us to hypothesize that Coa4 and Coa6 play redox roles in Cu delivery to CcO. We will test this hypothesis by determining the in vitro and in vivo redox states of Coa4- and Coa6- interacting proteins and by defining the physical basis of interaction through genetic and protein:protein interaction studies. Thus, the proposed project addresses fundamental questions in Cu delivery mechanisms to CcO.